www.ijper.org
Synthesis, Characterization, Molecular Docking
Studies and Antimicrobial Evaluation of
N-Benzimidazol-1-Yl-Methyl-Benzamide Derivatives
Ritchu Sethi1, Sandeep Arora1, Deepika Saini2 and Sandeep Jain2*
1Chitkara College of Pharmacy, Chitkara University, Rajpura, Distt. Patiala-140401, INDIA. 2Department of Pharmaceutical Sciences Guru Jambheshwar University, Hissar-125001, INDIA.
ABSTRACT
N-benzimidazol-1-yl-methyl-benzamide derivatives (3a-3x) were synthesized by Mannich reaction and evaluated for in vitro antimicrobial activity against Escherichia coli,
Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Candida albicans and
Aspergillus niger. The structures of novel target compounds were elucidated by spectral and analytical techniques. Among the synthesized derivatives, 3o N-[2-(2-chloro-phenyl)-benzimidazol-1-ylmethyl]-benzamide, 3q N-[2-(4-chloro-phenyl)-benzimidazol-1ylmethyl]-benzamide and 3r N-[2-(2-bromo-phenyl)-benzimidazol-1-ylmethyl]-benzamide were found to be most effective antimicrobial compounds. Clotrimazole and ciprofloxacin were used as reference antimicrobial agents. Further, in silico studies were carried out to define the interaction of the title compounds with microbial protein.
Key words: Mannich bases, Benzimidazole, Antibacterial activity, Antifungal activity, Docking study.
DOI: 10.5530/ijper.50.3.16
Correspondence:
Sandeep Jain,
Department of Pharma-ceutical Sciences, Guru Jambheshwar University of Science and Technology, Hissar, INDIA.
Mobile: +919416498857 E-mail: drsjain1969@yahoo. co.in
INTRODUCTION
Antimicrobial resistance is an evolving predicament in treating the patients, and causes several deaths every year.1 The main
cause of microbial resistance is the mutations or transfer of resistant genes between organisms.2 Resistant microbes may require
other medications or elevated doses associ-ated with more side effects, some of which may be life alarming. The chronic use of antibiotics is escalating the rates of infections, due to antibiotic resistance. As resistance to antibiotics becomes more common, the development of novel, effective and inimitable antimicrobial agents is the only superlative way to conquer microbial resistance and develop effective therapies.
Despite of several attempts to build up new structural analogues in the seek for more effective antimicrobials, the benzimidazoles
still remain the most flexible class of com
-pounds against microbes.3-5 2- substituted
benzimidazole and its derivatives have attracted great attention for the past few decades due to their chemotherapeutic val-ues and therefore, are useful compounds for further molecular discovery. These deriva-tives show biological activities such as anti-microbial,6 anti-allergic,7 PARP (poly ADP
ribose polymerase) inhibitors- as anticancer agents,8 as cytomegalovirus (HCMV)
inhibi-tors,9antiulcer,10 anti inflammatory,11 and as
antihistaminics.12
On the other side mannich bases are the end products of mannich reaction and are known as beta amino ketone carrying compounds.13 Mannich bases are very
reac-tive because of the introduction of basic functional group which renders the deriva-tive in aqueous solvent and can be changed into several other compounds.14 Literature
Submission Date : 14-03-2016
Revision Date : 12-05-2016
revealed that mannich bases have been reported to possess analgesic,15 anti-inflammatory,16,17 anticancer,18,19
anti-convulsant,20 antibacterial,21,22 antifungal,22,23 and several
other activities.
Keeping the above facts in mind and as part of continuation of our research on Mannich bases,24 we hereby report
the synthesis and antimicrobial evaluation of mannich bases of 2-substituted benzimidazole derivatives. Their
structural configuration was elucidated with the elemental
and spectral analysis. Synthesized novel compounds derived from mannich reaction were further evaluated for antimicrobial activity and then screened by in silico
method to study drug receptor interaction between novel compounds and microbial protein.
MATERIALS AND METHODS
Chemicals were procured from Sigma Aldrich, USA. Melting points of the synthesized compounds were determined by open capillary tubes and were uncor-rected. The purity of the synthesized compounds was determined by thin layer chromatography on precoated silica gel G plates with visualization by iodine vapours/ UV chamber. Infrared spectra (KBr pellets) were recorded on Perkin Elmer Spectrum FTIR spectropho-tometer. Proton NMR spectra were recorded on Bruker Avance II 400 NMR Spectrometer using DMSO-d6 as a solvent and TMS (tetra methyl silane) as internal standard
(chemical shift in δ ppm). Elemental analyses were carried
out using Eager Xperience CHN analyzer at Panjab University, Chandigarh. Mass spectra were taken on Q-TOF MICROMASS Spectrometer (WATERS, USA).
Chemistry
Mannich Bases of 2-substituted benzimidazole were synthesized by the reaction of 2-substituted benzimidazole (secondary amine), formalin and benzamide (active
hydrogen compound) (3a-x). 2-substituted benzimidazoles (2a-x) were prepared by the reaction of ortho-phenyl
-enediamine with substituted carboxylic acid and with
substituted aromatic aldehyde respectively.
General schemes for the synthesis of compounds
General method
The title compounds were prepared by the following steps.
Synthesis of 2-substituted benzimidazoles from ortho-phenylene-diamine dihydrochloride[2(a-f),2h,2j,2v]
2-substituted benzimidazoles were synthesized by the reaction of o-phenylenediamine dihydrochloride with
substituted carboxylic acid by the method described in
literature.25
Synthesis of 2-substituted benzimidazoles from ortho-phenylene-diamine [2(k-x),2i,2g]
2- substituted benzimidazoles were synthesized by the reaction of o-phenylenediamine and substituted benzal-dehyde by the procedure reported in literature.26-28 Synthesis of mannich base of 2-substituted benzimidazoles with benzamide [3(a-x)]
2- substituted benzimidazole (0.01 mole) was added to the ethanolic solution of benzamide (0.01mole). Form-aldehyde (37%) (0.01 mole) was added and the reaction
mixture was then adjusted to the pH of 3.5 with conc. HCl. The mixture was kept at efficient ice cooling for half an hour. Then it was refluxed with stirring at 80°C
for 10-12 hrs. Formaldehyde (37%) was added to the
reaction mixture in portions for completion of the reac -tion. End of reaction was monitored by TLC. Solvent system- CHCl3 : CH3OH ; 9.5: 0.5. Reaction mixture
was kept in refrigerator overnight. Solvent was evapo-rated under reduced pressure and product was collected, washed with water and recrystallized from ethanol. Similar type of procedures and equimolar quantities of reactants were used and 24 novel compounds were syn-thesized. Synthetic route for synthesis of novel com-pounds is shown in the general scheme. Physical data of synthesized compounds is given in Table 1.
Spectral data
N-(Benzimidazol-1-yl methyl)-benzamide. (3a)
White crystals, mp 198-199ºC, IR (KBr, cm-1) 3307 (N-H), 1486 (C=N), 3053 C-H(CH2), 2965 (C-H, Ar.) 1526 (C=C), 1634 (C=O), 675-870 (CH bend.Ar). 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.39-7.91 (m, 10H,
ArH), 4.98 (s, 2H, NCH2N), 8.70 (s, 1H, NH). Anal Calcd for C15H13N3O: C 71.70 , H 5.21, N 16.72 ; found C 70.84, H 5.14, N 15.94.
N-(2-methyl-benzimidazol-1-yl methyl)-benzamide (3b)
White crystals, mp 180-182ºC, IR (KBr, cm-1) 3308 (N-H), 1487 (C=N), 3055 C-H(CH2), 2966 (C-H, Ar.), 1526 (C=C), 1634 (C=O), 675-870 (CH bend.Ar). 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.3-7.9 (m, 9H,
ArH), 4.89 (s, 2H, NCH2N), 8.8 (s, 1H, NH), 2.80 (s, 3H, CH3) Anal Calcd for C16H15N3O: C 72.43, H 5.7, N 15.84; found C 69.84, H 5.54, N 15.69. MS: m/z = 266.2 (M+1).
N-(2-chloromethyl-benzimidazol-1-yl methyl)-benzamide (3c)
7.4-8.1 (m, 9H, ArH), 4.92 (s, 2H, NCH2N), 9.0 (s, 1H, NH), 5.19 (s, 2H, CH2). Anal Calcdfor C16H14ClN3O: C 64.11, H 4.71, N 14.02; found C 65.06, H 4.41, N 13.9. MS: m/z = 300.2 (M+1).
N-(2-ethyl-benzimidazol-1-yl methyl)-benzamide (3d)
White crystals, mp 210-212ºC, IR (KBr, cm-1) 3308 (N-H), 1486 (C=N), 3019 C-H(CH2), 2967 (C-H, Ar.) 1526 (C=C), 1634 (C=O), 675-870 (CH bend.Ar).
1HNMR(400 MHz, DMSO-d6, δppm), 7.3-7.9 (m, 9H,
ArH), 4.99 (s, 2H, NCH2N), 8.8 (s, 1H, NH), 3.27 (q, 2H, CH2, J=7.6 Hz), 1.56 (t, 3H, CH3, J=7.6 Hz). Anal Calcdfor C17H17N3O: C 73.10, H 6.13 N 15.04; found C 71.06, H 5.98, N 14.9. MS: m/z = 279.2 (M+1).
N-(2-propyl-benzimidazol-1-yl methyl)-benzamide (3e)
White crystals, mp 208-210ºC, IR (KBr,cm-1) 3308 (N-H), 1487 (C=N), 3056 C-H(CH2), 2967 (C-H,Ar.) 1527
(C=C), 1634 (C=O), 675-870 (CH bend.Ar). 1HNMR
(400MHz, DMSO-d6, δppm), 7.40-8.12 (m, 9H, ArH),
4.93 (s, 2H, NCH2N), 8.9 (s, 1H, NH), 2.96 (t, 2H, CH2,
J=7.6 Hz), 1.93 (sextet, 2H, CH2, J=7.6 Hz), 1.0 (t, 3H, CH3, J=7.2 Hz). Anal Calcdfor C18H19N3O: C 73.69, H 6.53, N 14.32; found C 75.4, H 5.96 N 14.15.
N-(2-butyl-benzimidazol-1-yl methyl)-benzamide (3f)
White crystals, mp 212-214ºC, IR (KBr, cm-1) 3307 (N-H), 1486 (C=N), 3055 C-H(CH2), 2966 (C-H, Ar.) 1527 (C=C), 1635 (C=O), 675-870 (CH bend.Ar). 1H NMR (400MHz, DMSO-d6, δppm), 7.18-8.06 (m, 9H,
ArH), 4.97 (s, 2H, NCH2N), 8.84 (s, 1H, NH), 0.93 (t, 3H, CH3, J=7.2 Hz), 1.40 (sextet, 2H, CH2, J=7.6 Hz), 1.82 (p, 2H, CH2, J= 2.16 Hz), 2.93 (t, 2H, CH2, J=7.6 Hz). Anal Calcd for C19H21N3O: C 74.24, H 6.89, N 13.67; found C 69.4, H 6.59, N 12.43.
Table 1: Physical data of synthesized compounds
Compound R Molecular formula Molecular weight Point(ºC)Melting Yield (%) Rf value
3a H- C15H13N3O 251.28 198-199 66.2 0.78
3b CH3- C16H15N3O 265.31 180-182 62.4 0.69
3c ClCH2- C16H14ClN3O 299.75 190-191 76.5 0.59
3d C2H5- C17H17N3O 279.34 210-212 64.2 0.64
3e C3H8- C18H19N3O 293.36 208-210 62.5 0.77
3f C4H10- C19H21N3O 307.39 212-214 60.6 0.91
3g NH2- C15 H14 N4 O 266.30 205-206 58.4 0.90
3h C17 H17 N3O2 295.34 190-192 56.2 0.65
3i SH- C15 H13 N3OS 283.35 170-172 52.7 0.91
3j SHCH2- C16 H15 N3 OS 297.38 212-214 51.8 0.84
3k C6H5- C21 H17 N3 O 327.38 195-197 80.4 0.95
3l (2-N-C5H4)- C20 H16 N4 O 328.37 218-220 72.5 0.77
3m (3-N-C5H4)- C20 H16 N4 O 328.37 205-206 79.7 0.85
3n (2-OH-C6H4)- C21 H17 N3 O2 343.38 180-182 75.3 0.84
3o (2-Cl-C6H4)- C21H16ClN3O 361.82 190-191 80.0 0.87
3p (3-Cl-C6H4)- C21H16ClN3O 361.82 200-202 71.3 0.86
3q (4-Cl-C6H4)- C21H16ClN3O 361.82 195-196 73.6 0.75
3r (2-Br-C6H4)- C21H16BrN3O 406.28 190-192 75.4 0.81
3s (3-Br-C6H4)- C21H16BrN3O 406.28 192-194 76.7 0.80
3t (4-Br-C6H4)- C21H16BrN3O 406.28 195-196 78.6 0.77
3u (4-NO2-C6H4)- C21H16N4O3 372.38 180-183 79.2 0.64
3v (-C6H5- CH2)- C22H19N3O 341.41 170-172 64.6 0.70
3w (4-F-C6H4)- C21H16FN3O 345.37 230-231 72.7 0.76
3x (2-NH2-C6H4)- C21H18N4O 342.39 206-208 78.5 0.88
Scheme 2: Synthesis of mannich bases from 2-substituted benzimidazoles.
Scheme 1a: Synthesis of 2-substituted benzimidazoles using
o-phenylene diamine dihydrochloride. Scheme 1b: Synthesis of 2-substituted benzimidazoles using o-phenylene diamine.
N-(2-amino-benzimidazol-1-yl methyl)-benzamide (3g)
Yellow crystals; mp. 205-206ºC, IR (KBr, cm-1) 3305 (N-H), 1484 (C=N), 3165 C-H(CH2), 3060 (C-H, Ar.) 1529 (C=C), 1638 (C=O), 675-870 (CH bend.Ar).1H NMR(400MHz, DMSO-d6, δppm), 7.21-7.96 (m, 9H,
ArH), 7.97 (s,1H, NH2), 4.90 (s, 2H, NCH2N), 9.03(s, 1H, NH), 9.6 (s, 1H, NH2). Anal Calcdfor C15 H14 N4 O: C 67.65, H 5.30 , N 21.04 ; found C 65.3, H 4.92, N 20.3.
N-2-[(1-hydroxy-ethyl)-benzimidazol-1-ylmethyl]-benzamide (3h)
White crystals, mp 190-192ºC IR (KBr, cm-1) 3308 (N-H), 1487 (C=N), 3056 C-H(CH2), 2967 (C-H, Ar.) 1527 (C=C), 1634 (C=O), 675-870 (CH bend.Ar).1H NMR(400MHz, DMSO-d6, δppm), 7.39-7.92 (m, 9H,
ArH), 4.90 (s, 2H, NCH2N), 8.97 (s, 1H, NH), 5.70 (br, s, 1H, OH), 1.59 (d, 3H CH3, J= 6.4 Hz), 5.03 (1H, q, CH, J= 6.8 Hz).Anal Calcdfor C17 H17 N3O2: C 69.14, H 5.80, N 14.23 ; found C 68.3, H 5.42, N 13.92.
N-(2-mercapto-benzimidazol-1-ylmethyl)-benzamide (3i)
White crystals, mp 270-272ºC, IR (KBr, cm-1) 3305 (N-H), 1378 (C=N), 3178 C-H(CH2), 3056 (C-H, Ar.) 1526 (C=C), 1635 (C=O), 675-870 (CH bend.Ar).1H NMR(400MHz, DMSO-d6, δppm), 7.26-7.95 (m, 9H,
ArH), 4.95 (s, 2H, NCH2N), 8.86 (s, 1H, NH), 3.4
(s, 1H, SH). Anal Calcdfor C15 H13 N3OS : C 63.58, H 4.62, N 14.83; found C 62.3, H 4.42, N 14.62.
N-(2-mercaptomethyl-benzimidazol-1-ylmethyl)-benzamide (3j)
White crystals, mp 212-214ºC, IR (KBr, cm-1) 3308 (N-H), 1487 (C=N), 3056 C-H(CH2), 2966 (C-H, Ar.) 1526 (C=C), 1634 (C=O), 675-870 (CH bend.Ar). 1H NMR (400MHz, DMSO-d6, δppm), 7.4-8.12 (m, 9H,
ArH), 4.92 (s, 2H, NCH2N), 8.98 (s, 1H, NH), 3.39 (s, 1H, SH), 4.18 (s, 2H, CH2). Anal Calcdfor C16 H15 N3 OS : C 64.62, H 5.08, N 14.13; found C 63.3, H 4.96, N 13.92. MS: m/z = 298.2 (M+1).
N-(2-phenyl-benzimidazol-1-ylmethyl)-benzamide (3k)
Brown crystals; mp. 195-197 ºC, IR (KBr, cm-1) 3308 (N-H), 1488 (C=N), 3056 C-H(CH2), 2965 (C-H, Ar.) 1526 (C=C), 1634 (C=O), 675-870 (CH bend.Ar). 1H NMR (400MHz, DMSO-d6, δppm), 7.4-8.32 (m, 14H,
ArH), 4.93 (s, 2H, NCH2N), 9.0 (s, 1H, NH). Anal Calcd for C21 H17 N3 O: C 77.04, H 5.23, N 12.84 ; found C 76.3, H 5.20, N 12.72. MS: m/z = 329.2 (M+1).
N-(2-pyridin-2-yl-benzimidazol-1-ylmethyl)-benzamide (3l)
Off white crystals; mp. 218-220ºC, IR (KBr, cm-1) 3308 (N-H), 1447 (C=N), 3056 C-H(CH2), 2967 (C-H, Ar.) 1527 (C=C), 1634 (C=O), 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6, δppm), 7.39-7.91 (m, 13H,
ArH), 4.9 (s, 2H, NCH2N), 8.8 (s, 1H, NH). Anal Calcd for C20 H16 N4 O: C 73.15, H 4.91, N 17.06 ; found C 73.3, H 4.20, N 16.72.
N-(2-pyridin-3-yl-benzimidazol-1-ylmethyl)-benzamide (3m)
Yellow crystals; mp. 205-206ºC, IR (KBr, cm-1) 3308 (N-H), 1486 (C=N), 3053 C-H(CH2), 2966 (C-H,Ar.), 1527 (C=C), 1635 (C=O), 675-870 (CH bend.Ar). 1H NMR (400MHz, DMSO-d6,δppm), 7.2-8.96 (m, 13H,
N-[2-(2-hydroxy-phenyl)-benzimidazol-1-ylmethyl]-benzamide (3n)
White crystals; mp. 180-182ºC, IR (KBr, cm-1) 3310 (N-H), 1491 (C=N), 3057 C-H(CH2), 2964 (C-H, Ar.) 1527 (C=C), 1633 (C=O), 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6,δppm), 7.04-8.18 (m, 13H,
ArH), 4.91 (s, 2H, NCH2N), 9.0 (s, 1H, NH), 13.6 (br,s, 1H, OH). Anal Calcdfor C21 H17 N3 O2: C 73.45 , H 4.99, N 12.24 ; found C 72.06, H 4.63, N 11.92. MS: m/z = 344.8 (M+1).
N-[2-(2-chloro-phenyl)-benzimidazol-1-ylmethyl]-benzamide (3o)
White crystals; mp. 190-191ºC, IR (KBr, cm-1) 3307 (N-H), 1485 (C=N), 3048 C-H(CH2), 2963 (C-H, Ar.) 1526 (C=C), 1634 (C=O), 770 (C-Cl), 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6, δppm),
7.41-8.9 (m, 13H, ArH), 4.91 (s, 2H, NCH2N), 9.01(s, 1H, NH). Anal Calcdfor C21H16ClN3O: C 69.71, H 4.46, N 11.61; found C 68.5, H 4.31, N 10.2. MS: m/z = 362.2 (M+1), 363(M+2).
N-[2-(3-chloro-phenyl)-benzimidazol-1-ylmethyl]-benzamide (3p)
White crystals; mp. 200-202ºC, IR (KBr, cm-1) 3309 (N-H), 1485 (C=N), 3034 C-H(CH2), 2953 (C-H,Ar.) 1527 (C=C), 1633 (C=O), 765 (C-Cl), 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6, δppm),
7.46-8.7 (m, 13H, ArH), 4.98 (s, 2H, NCH2N), 8.9 (s, 1H, NH). Anal Calcdfor C21H16ClN3O: C 69.71, H 4.46, N 11.61; found C 68.5, H 4.81, N 11.2.
N-[2-(4-chloro-phenyl)-benzimidazol-1-ylmethyl]-benzamide (3q)
White crystals; mp. 195-196ºC, IR (KBr, cm-1) 3307 (N-H), 1485 (C=N), 3045 C-H(CH2) 2952 (C-H, Ar.) 1525 (C=C), 1635(C=O), 764 (C-Cl) 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6, δppm),
7.35-8.6 (m, 13H, ArH), 4.96 (s, 2H, NCH2N), 8.90 (s, 1H, NH). Anal Calcd for C21H16ClN3O: C 69.71, H 4.46, N 11.61 ; found C 68.52, H 4.31, N 10.8. MS: m/z = 363.9 (M+2).
N-[2-(2-bromo-phenyl)-benzimidazol-1-ylmethyl]-benzamide (3r)
White crystals; mp. 190-192ºC, IR (KBr, cm-1) 3309 (N-H), 1488 (C=N), 3056 C-H(CH2), 2967 (C-H, Ar.) 1527 (C=C), 1635 (C=O), 869 (C-Br), 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6, δppm),
7.4-8.1 (m, 13H, ArH), 4.91(s, 2H, NCH2N), 8.9 (s, 1H, NH). Anal Calcdfor C21H16BrN3O: C 62.08, H 3.97, N 10.34; found C 63.3, H 2.98, N 9.43. MS: m/z = 406.2 (M+1), 407.3 (M+2).
N-[2-(3-bromo-phenyl)-benzimidazol-1-ylmethyl]-benzamide (3s)
White crystals; mp. 192-194ºC, IR (KBr, cm-1) 3307 (N-H), 1487 (C=N), 3034 C-H(CH2), 2966 (C-H, Ar.) 1525 (C=C), 1634 (C=O), 865 (C-Br), 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6, δppm),
7.1-8.7 (m, 13H, ArH), 4.98(s, 2H, NCH2N), 9.0 (s, 1H, NH). Anal Calcdfor C21H16BrN3O: C 62.08, H 3.97, N 10.34 ; found C 61.3, H 3.90, N 9.49.
N-[2-(4-bromo-phenyl)-benzimidazol-1-ylmethyl]-benzamide (3t)
Offwhite crystals; mp. 195-196ºC, IR (KBr, cm-1) 3308 (N-H), 1486 (C=N), 3056 C-H(CH2), 2967 (C-H, Ar.) 1527 (C=C), 1635 (C=O), 865(C-Br), 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6, δppm),
7.2-8.6 (m, 13H, ArH), 4.97 (s, 2H, NCH2N), 8.89 (s, 1H, NH). Anal Calcdfor C21H16BrN3O: C 62.08, H 3.97, N 10.34 ; found C 61.9, H 2.96, N 9.82.
N-[2-(4-nitro-phenyl)-benzimidazol-1-ylmethyl]-benzamide (3u)
Yellow crystals; mp. 180-183ºC, IR (KBr, cm-1) 3308 (N-H), 1463 (C=N), 3058 C-H(CH2), 2966 (C-H, Ar.) 1524 (C=C), 1634 (C=O), 747 (C-NO2), 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6, δppm),
7.24-8.46 (m, 13H, ArH), 4.94 (s, 2H, NCH2N), 8.90 (s, 1H, NH). Anal Calcd for C21H16N4O3: C 67.73, H 4.33, N 15.05; found C 66.3, H 4.28, N 14.96. MS: m/z = 373.7 (M+1).
N-(2-benzyl-benzimidazol-1-ylmethyl)-benzamide (3v)
White crystals; mp. 170-172ºC, IR (KBr, cm-1) 3308 (N-H), 1487 (C=N), 3054 C-H(CH2), 2966 (C-H, Ar.) 1525 (C=C), 1634 (C=O), 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6, δppm), 7.24-7.93 (m, 14H,
ArH), 4.93(s, 2H, NCH2N), 8.62 (s, 1H, NH), 4.53 (s, 2H, CH2). Anal Calcdfor C22H19N3O: C 77.40, H 5.61, N 12.31; found C 76.3, H 5.49, N 11.94.
N-[2-(4-flouro-phenyl)-benzimidazol-1-ylmethyl]-benzamide (3w)
White crystals; mp. 230-231ºC, IR (KBr, cm-1) 3310 (N-H), 1458 (C=N), 3055 C-H(CH2), 2927 (C-H, Ar.) 1525 (C=C), 1634 (C=O), 848 (C-F), 675-870 (CH bend. Ar). 1H NMR(400MHz, DMSO-d6, δppm), 7.4-8.48 (m,
13H, ArH), 4.90 (s, 2H, NCH2N), 9.03 (s, 1H, NH). Anal Calcdfor C21H16FN3O: C 73.03, H 4.67, N 12.17; found C 72.7, H 3.98, N 11.82. MS: m/z = 346.3 (M+1).
N-[2-(2-amino-phenyl)-benzimidazol-1-ylmethyl]-benzamide (3x)
1525 (C=C), 1634 (C=O), 675-870 (CH bend.Ar). 1H NMR(400MHz, DMSO-d6, δppm), 6.52-7.95 (m, 13H,
ArH), 4.98 (s, 2H, NCH2N), 8.79 (s, 1H, NH), 4.0 (s, 2H, NH2). Anal Calcdfor C21H18N4O: C 73.67, H 5.30, N 16.36; found C 71.9, H 4.96, N 15.82. MS: m/z = 343.5 (M+1).
Antimicrobial Evaluation
The synthesized compounds were screened for their
in vitro antimicrobial activity against two gram positive bacterial strains: Staphylococcus aureus (MTCC 96), Bacillus subtilis (MTCC 121), two gram negative bacterial strains:
Escherichia coli (MTCC 40), Pseudomonas aeruginosa (MTCC 2453), and two fungal strains: Candida albicans (MTCC 183) and Aspergillus niger (MTCC 404). The serial dilution technique was used for evaluation of antimicrobial activity. Stock solution of synthesized compounds was
prepared in dimethyl sulphoxide (100 µg/ml). Nutrient broth (I.P.) and sabouraud dextrose broth media (I.P.)29 were used for bacteria and fungi respectively. Ciprofloxacin
and clotrimazole were used as standard drugs for anti-bacterial activity and antifungal activity respectively. Sterlized media (1 ml) was transferred into sterile test
tubes. Stock solution (100 µg/ml) (1ml) was put in one
tube and serially diluted to give concentrations of 50,
25, 12.5, 6.25, 3.125 and 1.56 µg/ml. 0.1ml suspension
of bacteria and fungus in saline was added to all test tubes and were then incubated at 37 ± 1ºC for 24 hours
(bacteria) and 25ºC for 72 hrs (fungi). The test mixture
contained 106 organisms/ml (CFU/ml). Macroscopic examination of inoculated culture tubes was done for
turbidity. MIC was the lowest concentration at which microbial growth attenuated and no turbidity was seen in the tube. Same procedure was followed for reference
drugs and the experiment was done in triplicate. Molecular Docking studies
To get more insight into the binding interaction of the synthesized compounds, molecular docking studies were performed using Autodock Vina software. Marvin
Sketch application of ChemAxon was used for drawing
all the ligands employed in this study. The 3D structures of enoyl reductase-NAD+-triclosan complex (PDB id:
1C14) was procured from Protein Data Bank (www.rcsb. org).30 Before performing the docking, protein receptor
was prepared by merging all the non-polar hydrogens and removing the water of crystallization using the same graphical interface. Autodock tool uses the hybrid global-local search algorithm which is a big improvement
in the genetic algorithm for the best confirmations of legends. Then the protein was defined for the generation of active site, i.e. grid with specific dimensions. The parameters of grid box are given in Table 2. The
Autodock Vina uses a hybrid scoring function (empirical +
knowledge based function) for evaluating binding affinity
of ligands with the receptor.31
A set of 24 compounds was screened for antimicrobial activity by molecular docking simulations using PDB id : 1C14. The screening results were further compared with the in-vitro studies for their drug receptor interaction.
RESULTS AND DISCUSSION
Chemistry: In this study, 24 novel compounds
incor-porating benzimidazole scaffold were prepared and screened for antimicrobial activity. The reaction order for the synthesis of title compounds is given in general schemes. 2-substituted benzimidazoles were prepared by the reaction of ortho-phenylenediamine
dihydro-chloride with substituted carboxylic acid (Scheme 1a)
and by the reaction of ortho-phenylene diamine with substituted aromatic aldehyde, carbon disulphide and cynogen bromide (Scheme 1b). Then Mannich bases were prepared by the reaction of Benzamide (active hydrogen compound), secondary amine (2-substituted benzimidazole), formaldehyde and conc. hydrochloric acid (Scheme 2). The structures of all the compounds were elucidated by spectral analysis. From IR spectra, the appearance of peaks at 3308-3310 cm- and 1400-1500 cm- indicated the presence of NH of carboxamide and
C=N stretching of benzimidazole respectively. From NMR spectra, a 2 protons singlet at 4.8-4.99 ppm
confirmed the presence of CH2 attached to NH in all the synthesized compounds. The emergence of sharp singlet at 8.0-8.9 ppm ascertained the presence of NH
of carboxamide in all the synthesized compounds. The appearance of multiplet at 6.30-8.0 ppm confirmed
the presence of aromatic and hetero-aromatic protons. The calculated molecular weight of the synthesized compounds was comparable with observed m/e value. Hence, the calculated values of the projected
struc-tures were found to be in good confirmity with the data obtained experimently.
Antimicrobial Evaluation of test compounds: From
Table 3, it was found that amongst all synthesized com-pounds, 3o, 3q, 3r, 3t, 3u and 3w were most significantly
active against bacterial strains as compared to standard
ciprofloxacinand rest of the compounds showed moderate activity.
Structure activity relationship revealed that compound
3o with its electron withdrawing Cl group at 2 position of phenyl ring which is substituted at 2-position of benzimidazole showed better activity against P. auregenosa, S. aureus, B. subtilis and similar effect against E. coli than
elec-Table 2: x, y, z coordinates of grid box (PDB ID: 1C14)
center_x -1.723
center_y 32.185
center_z 145.55
size_x 40
size_y 40
size_z 40
tron withdrawing Cl group at 4 position of benzene ring, showed improved activity against P.auregenosa, S. aureus and similar effect against E. coli and B. subtilis as compared to reference drug ciprofloxacin. Compound
3r with its elecron withdrawing bromo group at 2 position of phenyl ring showed better results against P. aerugenosa and similar effect against all the rest of the three strains
when compared with ciprofloxacin. Compound 3t,3u,
3w with electron withdrawing Br, NO2 and F groups at 4 position of phenyl ring respectively showed better activity against P. auregenosa and B. subtilis than standard
drug ciprofloxacin.
From Table 4, it was found that amongst all the synthe-sized compounds, 3o, 3q and 3r with electron with-drawing Cl and Br groups at ortho and para positions of benzene ring respectively were most significantly
active against fungal strainsand rest of the compounds showed moderate activity as compared to standard clotrimazole. Hence, it is concluded that substitutions on heteroaromatic rings with electron withdrawing groups like Cl, Br, NO2, and F at 2 and 4 positions of phenyl rings showed enhanced antimicrobial activity as compared to reference drugs. No inhibitory effect was found for DMSO used as control.
Molecular Docking Studies: After in vitro antimicrobial evaluation, to understand the interaction of synthesized
Table 3: Minimum inhibitory concentration (MIC) (µg/ml) of the synthesized compounds against Gram positive and Gram negative bacteria
Compound (MTCC 40)E .coli (MTCC2453)P.aeruginosa (MTCC 96)S.aureus (MTCC121)B.subtilis
3a 12.5 12.5 12.5 12.5
3b 12.5 12.5 12.5 12.5
3c 6.25 12.5 12.5 12.5
3d 12.5 12.5 12.5 12.5
3e 12.5 12.5 12.5 12.5
3f 12.5 6.25 12.5 12.5
3g 12.5 12.5 12.5 12.5
3h 6.25 12.5 12.5 12.5
3i 12.5 6.25 12.5 6.25
3j 6.25 12.5 12.5 12.5
3k 6.25 6.25 12.5 12.5
3l 6.25 6.25 12.5 12.5
3m 12.5 12.5 6.25 12.5
3n 6.25 12.5 6.25 12.5
3o 3.125 6.25 3.125 3.125
3p 6.25 12.5 6.25 12.5
3q 3.125 6.25 3.125 6.25
3r 3.125 6.25 6.25 6.25
3s 12.5 6.25 12.5 12.5
3t 6.25 6.25 6.25 6.25
3u 6.25 6.25 3.125 6.25
3v 12.5 12.5 12.5 12.5
3w 6.25 6.25 12.5 3.125
3x 6.25 12.5 12.5 6.25
Ciprofloxacin 3.125 12.5 6.25 6.25
-Table 4: Minimum inhibitory concentration (MIC) (µg/ml) of the synthesized compounds against
fungal strains
Compound C. albicans
(MTCC 183) (MTCC 404)A.niger
3a 25 25
3b 25 25
3c 12.5 12.5
3d 25 25
3e 12.5 25
3f 25 25
3g 25 25
3h 25 25
3i 12.5 12.5
3j 25 25
3k 12.5 6.25
3l 12.5 6.25
3m 12.5 12.5
3n 6.25 12.5
3o 3.125 3.125
3p 6.25 12.5
3q 3.125 6.25
3r 6.25 3.125
3s 12.5 25
3t 6.25 6.25
3u 6.25 6.25
3v 25 25
3w 6.25 6.25
3x 6.25 12.5
Clotrimazole 6.25 6.25
Control -
-Figure 1: Binding pose for compound 3o (dock score -8.2 kcal/mol) within the domain of microbial receptor showing
hydrogen bonding in dashed green line.
Figure 2: Binding pose for compound 3r (dock score -7.9 kcal/mol) within the domain of microbial receptor showing
hydrogen bonding in dashed green line.
Figure 3: Binding pose for Standard Ciprofloxacin (dock score -6.6 kcal/mol) within the domain of microbial receptor
showing two hydrogen bonding in dashed green line.
Table 5: Data of dock score of synthesized compounds and interactive amino acids for antimicrobial activity
Comp R Dock score Number of hydrogen
bonds Amino acids involved in bonding
3a H- -7.0 -
-3b CH3- -7.2 -
-3c ClCH2- -7.0 1 Gln1040
3d C2H5- -6.5 -
-3e C3H8- -7.5
3f C4H10- -7.6 -
-3g NH2- -7.0 1 Gln1040
3h -6.8 -
-3i SH- -6.5 -
-3j SHCH2- -6.5 -
-3k C6H5- -7.7 1 Arg171
3l (2-N-C5H4)- -7.1 1 Gln40
3m (3-N-C5H4)- -7.3 1 Arg1171
3n (2-OH-C6H4)- -8.1 1 Arg171
3o (2-Cl-C6H4)- -8.2 1 Arg171
3p (3-Cl-C6H4)- -7.9 -
-3q (4-Cl-C6H4)- -8.1 -
-3r (2-Br-C6H4)- -7.9 1 Arg171
3s (3-Br-C6H4)- -7.8 1 Gln1040
3t (4-Br-C6H4)- -8.1 -
-3u (4-NO2-C6H4)- -8.0 -
-3v (-C6H5- CH2)- -7.9 -
-3w (4-F-C6H4)- -7.8 1 Arg171
3x (2-NH2-C6H4)- -8.1 1 Arg171
Internal ligand ciprofloxacin -6.6 2 Glu167, Val1244
compounds with microbial protein, in silico study was performed using molecular docking. Considering 1C14 as target, the series of compounds were docked to get the best in silico confirmations in the domain of 1C14 protein. Binding affinities of the synthesized compounds
were evaluated by using docking program autodock vina, which also optimised the antimicrobial activities of synthesized compounds as possible microbial inhibitors. During analysis, H-bonding and dock score were taken as two important parameters for obtaining the hits among the set of compounds. From the in silico study, it was revealed that the compounds were having higher
dock score than that of standard ciprofloxacin (-6.6
kcal/mol).The main amino acids which played a vital role in interaction are Gln1040, Arg171, Gln40, Arg 1171. Among the series, compound 3o has displayed best dock score of -8.2 kcal/mol with 1 H-bond, length
of 2.177 Ao, shown in Figure 1. Compound 3r also
displayed good dock score of -7.9 kcal/mol with 1 H bond, length of 2.204 Ao, shown in Figure 2. The data
of dock score and interactive amino acids of all the compounds of the series is provided in Table 5. The interactions of standard with the target protein and secondary structure of the receptor are displayed in Figure 3 and Figure 4 respectively. From the docking
study, it was confirmed that the compounds substituted
with halogen has displayed best interactions with the receptor among the series.
CONCLUSION
analysis. Synthesized compounds were then screened for in vitro antibacterial and antifungal activity. Also molecular docking studies were done and their inhibi-tory activity was tested against PDB ID: 1C14 micro-bial protein. Both the theory (Docking) and practice (MIC values) demonstrate that N-(Benzimidazol-1-yl methyl)-benzamide derivatives act as potent inhibi-tor of 1C14 microbial protein. All the synthesized derivatives showed momentous antimicrobial activity against bacterial strains E.coli (MIC 12.5-3.125 µg/
ml), P. aeruginosa (MIC 12.5-3.125 µg/ml), S.aureus (MIC 12.5-3.125 µg/ml) and B.subtilis (MIC
12.5-3.125 µg/ml) in comparison to reference drug cip
-rofloxacin and against fungal strains, C.albicans (MIC
25-3.125 µg/ml) and A. niger (MIC 25-3.125 µg/
ml) when compared with clotrimazole as standard antimicrobial agent. Amongst all the synthesized compounds, 3o, 3q, 3r, 3t, 3u and 3w with its elec-tron withdrawing substitutions (2-chloro, 4-chloro,
2-bromo, 4-bromo, 4-nitro and 4-flouro groups
respectively) on aromatic rings were the most active compounds against the bacterial strains. Compounds
3o and 3q with chloro substitution at 2 and 4
posi-tion of phenyl ring respectively and compound 3r
with its bromo substitution at 2 position of phenyl ring were the most active compounds against fungal strains. Hence it is concluded that N-(Benzimidazol-1-yl methyl)-benzamide derivatives with electron withdrawing substituents could be used for design-ing more potent antimicrobial agents. Further studies of these derivatives are highly suggested to judge the safety of novel compounds.
ACKNOWLEDGEMENT
The authors are thankful to Management, Chitkara Uni-versity, Punjab for providing necessary facilities to carry out this work.
CONFLICT OF INTEREST
The author declares that there is no conflict of interests.
ABBREVIATIONS USED
MS: Mass Spectrometry; FTIR: Fourier transform infrared spectroscopy; NMR: Nuclear magnetic reso-nance; IP: Indian Pharmacopoeia; TLC: Thin Layer Chromatography; DMSO-d6 : Deuterated Dimethyl
Sulphoxide; MIC: Minimum inhibitory concentration;
Int.lgn: Internal ligand; PDB: Protein Data Bank;
MTCC: Microbial Type culture collection.
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SUMMARY
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